Sarka Hruby

New monoclonal antibodies reactive with defined sequential epitopes in human myelin basic protein

Three new IgG monoclonal antibodies are described which recognise sequential epitopes of the human myelin basic protein (MBP) molecule in amino acid sequences 36-50, 64-75 and 80-89. Two of the secreting hybridomas were prepared by immunisation of mice with synthetic peptides. This procedure appears to generate antibodies of similar affinities to those made using intact myelin basic protein as the immunogen. It has the advantage that antibodies to preselected regions of the molecule can be made at will and the problem of subsequent epitope localisation is simplified. It is possible with synthetic peptides to generate antibodies of specificities which it would be impossible to achieve by immunisation with intact myelin basic protein. The monoclonal antibodies described here should be useful tools in studies of myelin catabolism in vivo and in vitro. Of particular interest is our Clone 22, making an antibody which reacts equally well with intact human MBP and synthetic peptide sequence 80-89 in liquid phase assays. Antibodies of this rare specificity have been claimed to be able to react with the peptides of myelin basic protein found in the spinal fluid of patients with multiple sclerosis.

Sites in Myelin Basic Protein that React with Monoclonal Antibodies

Abstract: The epitopes (antigenic sites) for seven monoclonal antibodies (MAbs) evoked in rats or mice by guinea pig or monkey myelin basic protein (BP) have been located in four different sequences of the BPs extracted from various species. Six of the MAbs were evoked by guinea pig BP. (1) One epitope, possibly a pair, is included within residues 1–14 of all BPs tested and reacts with two rat IgG MAbs. (2) A definite pair of over‐lapping epitopes includes the central Phe91‐Phe92 sequence. One epitope is contained entirely within sequence 90–99 and reacts with a rat IgG MAb. The substitution of Ser in chicken BP for Thr97 destroys this epitope. The other epitope appears to include residues on the amino side of Phe44 and even of His32 and suggests some tertiary structure in BP. This epitope reacts with a mouse IgM MAb that does not recognize the chicken substitution. (3) The third epitope lies within residues 114–121, specifically including Trp118, and reacts with a rat IgG MAb. A cross‐reacting epitope probably includes residues 44–45 in certain species (guinea pig and bovine but not rabbit). (4) Another pair of epitopes is located within residues 131–140 but is severely species‐restricted. This region in guinea pig BP evoked a species‐specific mouse IgM MAb. The same region in monkey BP evoked the seventh MAb, a mouse IgG, which reacts with human, chimpanzee, monkey, bovine, and rat‐18.5 kDa BPs and to a lesser extent rabbit BP but not with guinea pig, pig, or chicken BPs. Some tertiary structure in guinea pig BP is also suggested by the reactivities with the IgM MAb. All of the MAbs react with myelin in histologic preparations, but the optimum method of preparation of the tissue varies with each.

In vivo administration of anti-CD4 monoclonal antibody prolongs survival in longtailed macaques with experimental allergic encephalomyelitis

The in vivo administration of monoclonal antibody (mAb) to the CD4 antigen associated with helper T cells has been successful in prolonging the survival of nonhuman primates with experimental allergic encephalomyelitis (EAE). EAE was induced in 17 outbred longtailed macaques (Macaca fascicularis) by inoculation of homologous myelin basic protein (BP) in complete Freunds adjuvant (CFA). Treatment was begun at the onset of clinical signs. Eleven animals were treated with anti-CD4 mAb Leu3a (eight) or OKT4a (three). Of the six control animals, two received anti-CD8 mAb (Leu2a), and four were treated with saline. Specific T- and B-cell subsets which have been implicated in the development of EAE were monitored throughout the course of the disease by one- and two-color immunofluorescence (IF). The monkey anti-BP antibody and anti-mouse immunoglobulin (IgG) responses were measured by enzyme-linked immunoassay (ELISA) techniques, as were the levels of free-circulating murine IgG. The nature of the infiltrating lymphocytes in the brain was evaluated histologically post mortem. Our results indicate that anti-CD4 mAb can prolong survival and in some cases completely reverse the clinical appearance of the disease; however, relapses did occur. Treatments with Leu3a or OKT4a anti-CD4 mAbs reversed the ongoing depletion of CD4+ and CD8+ cells caused by the development of EAE and appeared to reduce the size and degree of inflammation in brain lesions. These treatments did not induce immunologic tolerance to mouse IgG since all of the anti-CD4-treated animals produced high titers of anti-mouse IgG antibodies. Treatment with Leu2a (anti-CD8) had no effect on the development of EAE. These results suggest that CD4+ cells are important to the pathogenesis of EAE in macaques and that manipulation of this subset with monoclonal antibodies may provide effective treatment of human demyelinating disease.

Fluctuations of T- and B-cell subsets in basic protein-induced experimental allergic encephalomyelitis (EAE) in long-tailed macaques☆

Experimental allergic encephalomyelitis (EAE) was induced in long-tailed macaques (Macaca fascicularis) by inoculation of autologous myelin basic protein (BP) in complete Freunds adjuvant. Natural killer (NK) cell activity and lymphocyte subsets detected by one- and two-color immunofluorescence were monitored longitudinally in these animals. A decrease in NK cell activity was detected at the onset of clinically defined disease. During the preclinical phase of EAE (5-7 days before the onset of clinical signs) the absolute number of T helper (CD4+) and T suppressor (CD8+) cells in the peripheral blood decreased significantly. Analysis of peripheral blood B cells revealed a selective depletion of IgD+ B cells and a corresponding increase in the number of IgD- B cells prior to and during the onset of clinical signs. Total B-cell numbers were not significantly different between EAE and normal groups. The increased proportion of IgD- B cells in BP-sensitized animals corresponded with the appearance of high titers of circulating anti-BP antibodies. Thus two-color analysis of B-cell subsets may be a sensitive indicator of B-cell activation and of abnormal immune status in EAE. Changes in lymphocyte subsets in macaques with EAE are compared with those in humans with multiple sclerosis.

Region-specific immunoassays for human myelin basic protein

Three monoclonal antibodies reactive with human myelin basic protein have been used to develop immunoradiometric assays for this protein. Clone 1, a mouse IgG2a, is reactive with an epitope in the region 129-138. Clone 2, a mouse IgG1, is reactive with the region 119-131. Clone 12, a rat IgG, is reactive with the region 86-96. Competition experiments show that the reactions of Clone 1 and Clone 2 are mutually exclusive, probably because of steric effects. In contrast, when either Clone 1 or Clone 2 react they cause minimal interference with the subsequent binding of Clone 12. Less than 1 ng/ml of myelin basic protein can be detected in each of the two immunoradiometric assays developed. Clone 12 can also be used on its own in a competitive immunoassay to detect around 2 ng/ml. Using an extraction technique before the assay, serum or plasma can also be investigated. Assays for defined regions of myelin basic protein should prove valuable in identifying the products of myelin catabolism in patients with demyelinating disease.

Evidence for specific polypeptide chain folding in myelin basic protein from reactions between fragments of the protein and monoclonal antibodies.

Abstract: The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the proteins polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe‐Phe (87–88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22–95 and 45–151, and lower, but significant, reactivity was shown by peptide 32–95. Only very weak reactivity was seen with peptide 44–95. No reactivity was observed with peptide 1–95 after its lysine residues were acetylated, acetamidinated, orguanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val‐Val‐His‐Phe‐Phe‐Lys‐Asn‐Ile‐Val (84–92). The specific conformation of this epitope, which includes probably the Lys‐89 and possibly the Asn‐90 and Val‐92 side chains, could be formed by the association of sequence 84–92 with either sequence Ile‐Leu‐Asp‐Ser‐Ile‐Gly‐Arg‐Phe‐Phe (37–45) or with sequence Val‐Leu‐Ser‐Arg‐Phe (108–112) to form p‐sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R. E. J. Neurochem. 46, 1612–1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species‐restricted sequence Arg‐Ala‐Asp‐Tyr‐Lys‐Ser‐Lys (129–135). The smallest available fragment that displayed full reactivity was peptide 22–164; peptides 45–167, 89–167, and 119–167, on the other hand, were only about one‐fourth as reactive as the intact protein. These results suggest that sequence 22–44 constitutes a region in peptide 22–164 and in the intact protein which stabilizes sequence 129–135 in the appropriate antibody‐binding conformation.

Monoclonal antibodies reactive with myelin basic protein

New monoclonal antibodies (MAbs) to myelin basic protein (BP) reveal epitopes to be in sequences 22-34, 75-82, 83-96, 118-131 and 125-131. Comparison of these results with those previously reported suggest that almost every sequence of about 10 amino acid residues may be sufficiently antigenic to make a single MAb but that certain regions are immunodominant, strong enough to make practically the same MAb repeatedly. One of these new MAbs (clone 3) has especially interesting reactivity, sharply limited to residues 75-82 in bovine and porcine BP: Lys-Ala-Gln-His-Gly-Arg-Pro. Whales presumably have the same sequence, since their BPs are fully reactive with clone 3 MAb, but all other species of BP, with known sequences of BP, have at least two changes in this sequence. Deletion of Lys75 (as in a tryptic peptide of porcine BP) reduces reactivity with the MAb about 10-fold, whereas substitution of Ala76 by Ser (as in all other species of BP) and either deletion of Gln77 (as in human, monkey and rabbit BP) or His78 (as in the guinea pig and rat BP) or substitution of Pro82 by Thr (as in human, monkey, rat and mouse BP) eliminates reactivity. We speculate that woodchuck and prairie dog BPs in this region closely resemble chicken BP, which has about 2% of the original reactivity. However, squirrel BP is unique, probably having only one of the changes in this region of BP, since it possesses 10-20 times the reactivity of chicken BP but still only 20-50% of the original reactivity with clone 3 MAb, a degree of reactivity not seen with any other species of BP.

Monoclonal Antibodies to Myelin Basic Protein

Abstract Two new monoclonal antibodies to myelin basic protein (BP) have been prepared. In contrast to the first reported (11), which is an IgG 1 directed against residues 130-137 of certain species, both of the new ones are IgM. One is directed against the region including Phe-Phe residues 90-91, a region which is relatively invariant among many species. The other is relatively species-restricted and probably directed against the guinea pig equivalent of residues 130-137. These antibodies should become useful reagents not only in preparing specific peptide fragments of BP but also in defining some of the elements in the catabolism of myelin in multiple sclerosis (MS), experimental allergic encephalomyelitis (EAE) and other diseases of the central nervous system.

Modulation of a Subset of CD4+ (T4) “T Helper” Cells in Active Multiple Sclerosis (MS) and Experimental Allergic Encephalomyelitis (EAE) in Macaquesa

A number of studies have suggested that CD4+ (T4, Leu3a) T helper/inducer (Th) cells play a role in the pathogenesis of MS. Lesion progression in MS is associated with large numbers of CD4+ cells a t the lesion margins.’ Similarly, a selective migration of Lyt-l+ cells into the C N S from the peripheral blood has been detected in mice developing EAE.2 Our own analysis of patients with active MS has revealed a selective loss of a subset of Th cells that is detectable only by two-color fluorescence-activated cell sorter a n a l y ~ i s . ~ Using monoclonal antibodies (MAb) to the CD4+ T-cell subset marker and the common leukocyte marker p220, CD4+ cells can be divided into two subsets: p220+ and p220-. We have measured the frequencies of these two subsets both in patients with MS and in long-tailed macaques (M. fascicularis) developing EAE. Chronic EAE was induced in macaques by inoculating animals with homologous myelin basic protein (BP) in complete Freund’s adjuvant according to the method of Alvord et al.4 Blood was drawn once a week for complete blood counts and FACS cell sorter analyses of lymphocyte subsets. A comparison of the frequencies of p220tCD4+ cells and p220-CD4+ cells revealed that the p220+ subset was depleted in both patients with active MS and in animals induced to develop EAE. The p220+CD4+ cells were not depleted in patients with inactive MS or other neurologic diseases. Of ten animals inoculated with BP, six developed overt clinical signs and four developed subclinical EAE (TABLE 1). All of the animals with clinically active EAE had a selective depletion of p220+CD4+ cells. Two of four animals with subclinical EAE also had a loss of p220+CD4+ cells. This specific loss of p220+ cells is of interest because the p220 molecule is thought to play a role in